HIGH THROUGHPUT CALI FOR TARGET VALIDATION IN METASTASIS

转移是癌症最具破坏性的方面之一。因此， 发现抑制转移的治疗方法是一个重要的目标 癌症治疗。为了做到这一点，参与癌症的蛋白质 可能成为治疗药物靶点的细胞侵袭性必须是 经过验证，这通常是药物发现中的一个限速步骤。这个 总体目标是开发增强型高通量屏幕(HTS) 生色团辅助激光灭活(CALI)用于靶标 对在癌细胞侵袭中起作用的表面蛋白的验证。 CALI使用染料标记的抗体来灭活激光能量 结合抗原的功能。在R21阶段，HTS Transwell分析 将被开发和使用，以表明CALI将显著增加 破坏侵袭性的噬菌体展示抗体的数量。单人 与表面特异结合的链状FV融合噬菌体文库 将产生并使用人纤维肉瘤细胞HT-1080的蛋白质 对于使用CALI的试点筛查(n=96个抗体)。取得的成就 这些目标将确定HTS使用CALI用于 与癌细胞侵袭性有关的靶点的确认。在R33公路上 阶段，将进行全尺寸自动筛查(n=10,000 抗体)和蛋白质靶标将通过此筛选得到验证 使用高分辨率质谱仪进行鉴定。这项建议 技术将提供一种低成本和快速的目标验证手段 并将为抗转移药物贡献一系列靶点 发现号。开发的方法是通用的；它们将具有 应用于癌症和其他疾病的细胞过程。因此， 它们将对制药公司和学术界有很大的实用价值 实验室。

Metastasis is one of the most devastating aspects of cancer. Thus, discovering therapies that inhibit metastasis is an important goal in cancer treatment. In order to do this, proteins involved in cancer cell invasiveness that could be targets for therapeutic drugs must be validated and this is often a rate-limiting step in drug discovery. The overall objective is to develop an enhanced high throughput screen (HTS) using chromophore assisted laser inactivation (CALI) for target validation of surface proteins that act in cancer cell invasiveness. CALI targets laser energy using a dye-labeled antibody to inactivate the function of the bound antigen. In the R21 phase, an HTS Transwell assay will be developed and used to show that CALI will increased markedly the number of phage display antibodies that disrupt invasiveness. Single chain Fv fusion phage libraries that bind specifically to surface proteins of HT-1080 human fibrosarcoma cells will be generated and used for a pilot screen (n=96 antibodies) using CALI. The achievement of these aims will establish the feasibility of an HTS using CALI for the validation of targets involved in cancer cell invasiveness. In the R33 phase, a full scale automated screen will be conducted (n= 10,000 antibodies) and protein targets validated by this screen will be identified using high resolution mass spectrometry. This proposed technology will provide a low cost and rapid means of target validation and will contribute a number of targets for anti-metastasis drug discovery. The methods developed are general; they will have an application to cell processes in cancer and other diseases. As such, they will be of great utility for pharmaceutical companies and academic labs.